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Escherichia coli as a versatile cell factory: Advances and challenges in recombinant protein production Protein Expres. Purif. (IF 1.6) Pub Date : 2024-03-12 İbrahim İncir, Özlem Kaplan
plays a substantial role in recombinant protein production. Its importance increased with the discovery of recombinant DNA technology and the subsequent production of the first recombinant insulin in . is a widely used and cost-effective host to produce recombinant proteins. It is also noteworthy that a significant portion of the approved therapeutic proteins have been produced in this organism Despite
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Genetically encoded site-specific 19F unnatural amino acid incorporation in V. natriegens for in-cell NMR analysis Protein Expres. Purif. (IF 1.6) Pub Date : 2024-03-07 Hao Li, Jin Zhang, Zilong Wang, Pan Shi, Chaowei Shi
Nuclear magnetic resonance (NMR) spectroscopy NMR is a well-established technique for probing protein structure, dynamics and conformational changes. Taking advantage of the high signal sensitivity and broad chemical shift range of F nuclei, F NMR has been applied to investigate protein function at atomic resolution. In this report, we extend the unnatural amino acid site-specific incorporation into
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Unravelling the reason for different binding behaviors exhibited by antibody aggregates towards preparative and analytical Protein A columns Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-27 Wanyuan Dong, Mengying Tian, Yifeng Li
We previously showed that the root cause of low Protein A step yield observed for certain antibodies/Fc-fusions is the presence of non-binding aggregates in cell culture harvest. A pre-assumption for the above conclusion is that the aggregates, while do not bind to the preparative Protein A column, can bind to the analytical Protein A-high performance liquid chromatography (HPLC) column used for titer
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Chagasin from Trypanosoma cruzi as a molecular scaffold to express epitopes of TSA-1 as soluble recombinant chimeras Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-27 Rosa Elena Cárdenas-Guerra, Octavio Montes-Flores, Edgar Ezequiel Nava-Pintor, Gerardo Reséndiz-Cardiel, Claudia Ivonne Flores-Pucheta, Yasmín Irene Rodríguez-Gavaldón, Rossana Arroyo, Maria Elena Bottazzi, Peter J. Hotez, Jaime Ortega-López
is the causative agent of Chagas disease, a global public health problem. New therapeutic drugs and biologics are needed. The TSA-1 recombinant protein of is one such promising antigen for developing a therapeutic vaccine. However, it is overexpressed in as inclusion bodies, requiring an additional refolding step. As an alternative, in this study, we propose the endogenous cysteine protease inhibitor
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Effective expression and characterization of the receptor binding domains in SARS-CoV-2 Spike proteins from original strain and variants of concern using Bombyx mori nucleopolyhedrovirus in silkworm Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-22 Akira Tsukamoto, Lee Jae Man, Kosuke Oyama, Akitsu Masuda, Hiroaki Mon, Tadashi Ueda, Takahiro Kusakabe
A new coronavirus, known as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is responsible for the global pandemic of COVID-19 in 2020. Through structural analysis, it was found that several amino acid residues in the human angiotensin-converting enzyme-2 (hACE2) receptor directly interact with those in the receptor binding domain (RBD) of the spike glycoprotein (S-protein). Various cell
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Adapting recombinant bacterial alkaline phosphatase for nucleotide exchange of small GTPases Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-22 Peter Frank, Min Hong, Brianna Higgins, Shelley Perkins, Troy Taylor, Vanessa Wall, Matthew Drew, Timothy Waybright, William Gillette, Dominic Esposito, Simon Messing
The small GTPase Rat sarcoma virus proteins (RAS) are key regulators of cell growth and involved in 20–30% of cancers. RAS switches between its active state and inactive state via exchange of GTP (active) and GDP (inactive). Therefore, to study active protein, it needs to undergo nucleotide exchange to a non-hydrolysable GTP analog. Calf intestine alkaline phosphatase bound to agarose beads (CIP-agarose)
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Surface display system of Bacillus subtilis: A promising approach for improving the stability and applications of cellobiose dehydrogenase Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-17 Zhengfen Wu, Pengfei Li, Xihua Chen, Yong Feng, Yi Ma, Zhong Ni, Daochen Zhu, Huayou Chen
Cellobiose dehydrogenase (CDH) plays a crucial role in lignocellulose degradation and bioelectrochemical industries, making it highly in demand. However, the production and purification of CDH through fungal heterologous expression methods is time-consuming, costly, and challenging. In this study, we successfully displayed CDH (CDH) on the surface of spores for the first time. Enzymatic characterization
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Backbone cyclization of Salmonella typhimurium diaminopropionate ammonia-lyase to enhance the activity and stability Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-16 Xiaomei He, Tingting Lin, Yuying Xie, Jinjing Li, Yuanyuan Ge, Shuncheng Zhang, Jun Fan
Diaminopropionate ammonia-lyase transforms D and L isomers of 2,3-diaminopropionate to pyruvate and ammonia. It catalyzes D- and -serine less effectively. L-2,3-diaminopropionate is a precursor in the biosynthesis of oxalyl diaminopropionate as a neurotoxin in certain legume species. In this work, we cyclized the diaminopropionate ammonia-lyase from in vitro using the redox-responsive split intein
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High performance production process development and scale-up of an anti-TSLP nanobody Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-16 Xiaofei Li, Peng Qiao, Yicai Zhang, Guoxin Liu, Min Zhu, Junwei Gai, Yakun Wan
Nanobodies (Nbs) represent a class of single-domain antibodies with great potential application value across diverse biotechnology fields, including therapy and diagnostics. Thymic Stromal Lymphopoietin (TSLP) is an epithelial cell-derived cytokine, playing a crucial role in the regulation of type 2 immune responses at barrier surfaces such as skin and the respiratory/gastrointestinal tract. In this
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Novel melanin-derived stationary phase for immobilized metal ion affinity chromatography in recombinant His-tagged protein purification Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-14 Hong-Nhung Le Thi, Ngoc-Tram Le, Thu-Hoai Bui Thi, Hong-Loan Nguyen Thi, Thanh-Thuy Nguyen, Yen Nguyen Thi, Minh-Ngoc Ha, Dinh-Thang Nguyen
The matrix of the stationary phase is a crucial element in affinity chromatography for protein purification. Various materials, including polymer or magnetic materials, have been employed as the matrix in the purification of His-tagged protein. Here, for the first time, we utilized a combination of melanin and alginate, both natural polymer materials, to synthesize Ni-melanin/alginate (Ni-M/A) beads
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The expression of HIV-1 tat in Lactococcus lactis Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-14 Deepak Selvam, Anish D'silva, Arun Panchapakesan, Yuvrajsinh Gohil, Jayendra Singh, Luke Elizabeth Hanna, Udaykumar Ranga
Efficient expression of functional proteins in heterologous hosts has become the pivotal focus of modern biotechnology and biomedical research. To this end, multiple alternatives to are being explored for recombinant protein expression. , being a gram-positive organism, circumvents the need for an endotoxin removal step during protein purification. We report here the optimisation of the expression
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Evaluation of the paired-Cas9 nickase and RNA-guided FokI genome editing tools in precise integration of an anti-CD52 bicistronic monoclonal antibody expression construct at Chinese hamster ovary cells 18S rDNA locus Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-09 Hadi Bayat, Faranak Farahmand, Sayed Hassan Tabatabaee, Forough Shams, Omid Mohammadian, Es'hagh Pourmaleki, Azam Rahimpour
The aim of this study was to compare two CRISPR/Cas9-based orthogonal strategies, paired-Cas9 nickase (paired-Cas9n) and RNA-guided FokI (RFN), in targeting 18S rDNA locus in Chinese hamster ovary (CHO) cells and precisely integrating a bicistronic anti-CD52 monoclonal antibody (mAb) expression cassette into this locus. T7E1 and high-resolution melt (HRM) assays were used to compare the ability of
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Scalable dual column cation exchange affinity chromatography based platform process for recombinant protein purification Protein Expres. Purif. (IF 1.6) Pub Date : 2024-02-07 Sai Vivek Prabhala, David W. Wood
A novel tandem affinity tag is presented that enables the use of cation exchange resins for initial affinity purification, followed by an additional column step for enhanced purity and affinity tag self-removal. In this method, the highly charged heparin-binding tag binds strongly and selectively to either a strong or weak cation exchange resin based on electrostatic interactions, effectively acting
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Purification and characterization of an α-l-arabinofuranosidase, α-l-AFase, for hydrolyzed ginsenoside Rc from Bacillus subtilis Protein Expres. Purif. (IF 1.6) Pub Date : 2024-01-15 Ling Zhu, Qingfang Xu, Weiliang Liu, Qihe Xu, Lifang Zhang, Xiu Gao, Jian Cai
Natural ginsenoside needs to be converted into rare ginsenoside before it can be readily absorbed into the bloodstream for action. In this study, an α-l-arabinofuranosidase (α-l-AFase) gene Bsafs2 was cloned from Bacillus subtilis (B. subtilis). Bsafs2 was ligated to the expression vector pET28a(+), and the expression vector was constructed and transformed into Escherichia coli (E. coli) BL21 heterologous
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Recombinant protein production and functional analysis of a M60-like-2 metallopeptidase enzyme from the carcinogenic liver fluke Opisthorchis viverrini Protein Expres. Purif. (IF 1.6) Pub Date : 2024-01-06 Jiraporn Jumpajan, Sujittra Chaiyadet, Prasert Saichua, Sirikachorn Tangkawatana, Krajang Talabnin, Thewarach Laha, Sutas Suttiprapa
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Generation and characterization of nanobodies targeting human pepsinogens Protein Expres. Purif. (IF 1.6) Pub Date : 2024-01-05 Shenglan Zhang, Jieyao Ma, Liu He, Qianying Li, Pan He, Jing Li, Huicong Zhang
Human pepsinogens (mainly pepsinogen I and pepsinogen II) are the major inactive precursor forms of the digestive enzyme pepsin which play a crucial role in protein digestion. The levels and ratios of human pepsinogens have demonstrated potential as diagnostic biomarkers for gastrointestinal diseases, particularly gastric cancer. Nanobodies are promising tools for the treatment and diagnosis of diseases
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Expression, purification, characterization and crystallization of Panax quinquefolius ginsenoside glycosyltransferase Pq3-O-UGT2 Protein Expres. Purif. (IF 1.6) Pub Date : 2024-01-04 Qiushuang Ji, Yirong Liu, Cheng Chen, Huanyu Zhang, Juan Wang, Kunrong Mei
Pq3-O-UGT2, derived from Panax quinquefolius, functions as a ginsenoside glucosyltransferase, utilizing UDP-glucose (UDPG) as the sugar donor to catalyze the glycosylation of Rh2 and F2. An essential step in comprehending its catalytic mechanism involves structural analysis. In preparation for structural analysis, we expressed Pq3-O-UGT2 in the Escherichia coli (E. coli) strain Rosetta (DE3). The recombinant
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SEC-HPLC analysis of column load and flow-through provides critical understanding of low Protein A step yield Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-21 Chen Liu, Mengying Tian, Wanyuan Dong, Wenwen Lu, Ting Zhang, Yan Wan, Xudong Zhang, Yifeng Li
For a certain number of mAbs, bispecific antibodies (bsAbs) and Fc-fusion proteins that we worked on, the Protein A capture step experienced low yield (i.e., ∼80%). A previous case study suggested that non-binding aggregate formed in cell culture was the root cause of low Protein A step yield. In the current work, we selected five projects with the low Protein A yield issue to further illustrate this
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Expression and characterization of a lipase EstA from Bacillus subtilis KM-BS for application in bio-hydrolysis of waste cooking oil Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-16 Vinh D.H. Nguyen, Trang N.P. Huynh, Thao T.T. Nguyen, Hai H. Ho, Ly T.P. Trinh, Anh Q. Nguyen
A lipase EstA from Bacillus subtilis KM-BS was expressed in Escherichia coli BL21 (DE3) cells. The recombinant enzyme achieved high activity (49.67 U/mL) with protein concentration of 1.29 mg/mL under optimal conditions at the large-scale expression of 6 h and post-induction time at 30 °C using 0.1 mM isopropyl-β-d-thiogalactopyranoside (IPTG). The optimal temperature and pH of the purified enzyme
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Evaluate the potential use of TonB-dependent receptor protein as a subunit vaccine against Aeromonas veronii infection in Nile tilapia (Oreochromis niloticus) Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-15 Pornpavee Saichuer, Prit Khrisanapant, Saengchan Senapin, Triwit Rattanarojpong, Wasusit Somsoros, Pongsak Khunrae, Pakkakul Sangsuriya
Aeromonas veronii is an emerging bacterial pathogen that causes serious systemic infections in cultured Nile tilapia (Oreochromis niloticus), leading to massive deaths. Therefore, there is an urgent need to identify effective vaccine candidates to control the spread of this emerging disease. TonB-dependent receptor (Tdr) of A. veronii, which plays a role in the virulence factor of the organism, could
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Purification of thioredoxin reductase from Spirulina platensis by affinity chromatography and investigation of kinetic properties Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-16 Eda Dagsuyu, Refiye Yanardag
The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and nicotinamide adenine dinucleotide phosphate (NADPH). Spirulina platensis, which is one of the blue-green algae in the form of spiral rings, belongs to the cyanobacteria class. Spirulina platensis can produce Trx under stress conditions. If it can produce Trx, it also has TrxR activity. Therefore, in this study, the
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Cloning and expression of Neurospora crassa cellobiohydrolase II in Pichia pastoris Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-15 Jingsong Jia, Yu Tan, Jialun Gao, Jingjing Han, Peng Shi, Hao Fang, Chen Zhao
A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity
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Functional characterization and structural insights of three cutinases from the ascomycete Fusarium verticillioides Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-15 Caroline Torres de Oliveira, Michelle Alexandrino de Assis, João Paulo Lourenço Franco Cairo, André Damasio, Gonçalo Amarante Guimarães Pereira, Marcio Antonio Mazutti, Débora de Oliveira
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Improved expression and purification of highly-active 3 chymotrypsin-like protease from SARS-CoV-2 Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-09 Hong-Loan T. Nguyen, Nhu-Quynh T. Nguyen, The-Thai Le, Xuan-Dieu T. Pham, Hai-Long Pham, Hong-Nhung T. Le, Tuan-Nghia Phan, Nho-Thai Dinh
Severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is the causative pathogen of coronavirus disease-19 (COVID-19). The COVID-19 pandemic has resulted in millions of deaths and widespread socio-economic damage worldwide. Therefore, numerous studies have been conducted to identify effective measures to control the spreading of the virus. Among various potential targets, the 3 chymotrypsin-like
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Development of a bispecific Nanobody anti-F17 fimbria as a potential therapeutic tool Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-05 Asma Dhehibi, Mohammed Terrak, Mabrouk-Mouldi Seddik, Mohamed Hammadi, Imed Salhi
Pathogenic strains of Escherichia coli F17+ are associated with various intestinal and extra-intestinal pathologies, including diarrhea, and result in significant animal mortality. These infections rely on the expression of virulence factors, such as F17 fimbriae, for adhesion. F17 fimbriae form a protective layer on the surface of E. coli bacteria, consisting of a major structural subunit, F17A, and
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Prokaryotic expression of soluble IFN-λ1 recombinant protein with cold-shock system Protein Expres. Purif. (IF 1.6) Pub Date : 2023-12-07 Leiying Ren, Xueqiong Meng, Jie Sun, Xiaoya Shao, Mengyu Shao, Shuo Wang, Zhitao Li, Yixiang Chen
Interferon (IFN)-λ1, a member of type III IFN, possesses unique antiviral, anti-tumor, and immune modulation properties. IFN-λ alone or combined with other drugs is considered an essential therapeutic regimen in the clinic. Obtaining high-quality, biologically-active recombinant human IFN-λ1 (rhIFN-λ1) is of great practical significance. In this study, pCold–II–IFN-λ1 expression plasmid was correctly
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Recombinant expression of Yersinia ruckeri outer membrane proteins in Escherichia coli extracellular vesicles Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-30 Verena Mertes, Athanasios Saragliadis, Elisa Mascherin, Ellen-Beate Tysvær, Norbert Roos, Dirk Linke, Hanne C. Winther-Larsen
The secretion of extracellular vesicles (EVs) is a common process in Gram-negative bacteria and can be exploited for biotechnological applications. EVs pose a self-adjuvanting, non-replicative vaccine platform, where membrane and antigens are presented to the host immune system in a non-infectious fashion. The secreted quantity of EVs varies between Gram-negative bacterial species and is comparatively
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Fusion expression, purification, and characterization of cytokeratin 19 fragments in E. coli for enhanced stability in diagnostic applications Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-29 Yunbo Liu, Pan Zhu, Chengdong Ji, Lichun Dong, Weijing Yi
Cytokeratin 19 fragment (CYFRA21-1) serves as a crucial tumor marker in the context of lung cancer patients, playing a pivotal role as a calibrator in the realm of in vitro diagnostics. Nevertheless, during practical application, it has come to light that the recombinantly synthesized full-length CYFRA21-1 antigen exhibits suboptimal stability at the requisite concentration, while the utilization of
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Discovery and biochemical characterization of two hexokinases from Crassostrea gigas Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-24 Huibo Song, Bo Wang, Guihong Zhao, Shihai Lu, Dahu Zhang, Jianbiao Kong, Jianxin Li, Xiaoyang Zhang, Yongmei Lyu, Li Liu
Hexokinases (HKs) play a vital role in glucose metabolism, which controls the first committed step catalyzing the production of glucose-6-phosphate from glucose. Two HKs (CGIHK1 and CGIHK2) from the Pacific oyster Crassostrea giga were cloned and characterized. CGIHK1 and CGIHK2 were recombinantly expressed in Escherichia coli and successfully purified by the Ni-NTA column. The optimum pH of the two
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Cell-free protein production of a gamma secretase homolog Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-22 Celine Moser, Claudia Muhle-Goll
Cleavage of the transmembrane domain (TMD) of amyloid-β precursor protein (APP) by γ-secretase, an intramembrane aspartyl protease, generates Aβ peptides of various lengths that form plaques in the brains of Alzheimer's disease patients. Although the debate has not been finally resolved whether these plaques trigger the onset of Alzheimer's or are side products, disease-related mutations suggest their
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A streamlined, automated workflow to screen and triage large numbers of baculoviruses for protein expression Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-21 James J. Kostas, Andrea T. Partridge, Noel J. Byrne, Richard W. Edwards, Hua-Poo Su, Sandra B. Gabelli, Alexei Brooun, Jennifer M. Shipman
The baculovirus expression system is a powerful and widely used method to generate large quantities of recombinant protein. However, challenges exist in workflows utilizing either liquid baculovirus stocks or the Titerless Infected-Cells Preservation and Scale-Up (TIPS) method, including the time and effort to generate baculoviruses, screen for protein expression and store large numbers of baculovirus
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Evaluation of different strategies to produce Vibrio cholerae ParE2 toxin Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-17 Yana Girardin, Margot Galle, Yaël Vanden Abeele, Henri De Greve, Remy Loris
Toxin-antitoxin (TA) systems are small operons that are omnipresent in bacteria and archaea with suggested roles in stabilization of mobile genetic elements, bacteriophage protection, stress response and possibly persister formation. A major bottleneck in the study of TA toxins is the production of sufficient amounts of well-folded, functional protein. Here we examine alternative approaches for obtaining
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α-Conotoxin recombinant protein ImI-AFP3 efficiently inhibits the growth and migration of lung cancer cells Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-17 Xiaobing Chen, Zijuan Zou, Wei Li, Xu Dong, Yi Chen, Yan Lu, Mingyue Zhu, Mengsen Li, Bo Lin
α-Conotoxin ImI is a selective antagonist of alpha7 nicotinic acetylcholine receptor (α7 nAChR) that is involved in cancer development. Human alpha fetoprotein domain 3 (AFP3) is a prototype of anticancer agents. In an effort to design drugs for anticancer treatments, we fused the ImI peptide to AFP3 as a fusion protein for testing. The fusion protein (ImI-AFP3) was highly expressed in the insect Bac-to-Bac
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Cytoplasmic production of Fabs in chemically defined media in fed-batch fermentation Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-16 Angel Castillo-Corujo, Mirva J. Saaranen, Lloyd W. Ruddock
Fragment of antigen-binding region (Fab) of antibodies are important biomolecules, with a broad spectrum of functionality in the biomedical field. While full length antibodies are usually produced in mammalian cells, the smaller size, lack of N-glycosylation and less complex structure of Fabs make production in microbial cell factories feasible. Since Fabs contain disulfide bonds, such production is
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Acidic pH or salt treatment can convert soluble antibody aggregates in culture harvest into monomers and improve Protein A chromatography step yield Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-07 Ting Zhang, Bin Li, Wanyuan Dong, Yan Wan, Yifeng Li
While purifying a regular monospecific antibody, we found that the Protein A step yield was much lower than expected. Further studies revealed that the antibody formed large-size aggregates that did not bind to the Protein A resin, hence leading to dropped recovery. In an attempt to solve this low yield issue, we found that mildly acidic pH or ammonium sulfate treatment can partially convert the aggregates
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Enzymatic and antibacterial activity of the recombinant endolysin PVP-SE1gp146 expressed in Hansenula polymorpha Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-11 Abolghasem Asadi-Saghandi, Mojgan Bandehpour, Ali Hashemi, Bahram Kazemi
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A transmembrane scaffold from CD20 helps recombinant expression of a chimeric claudin 18.2 in an in vitro coupled transcription and translation system Protein Expres. Purif. (IF 1.6) Pub Date : 2023-11-10 Yao Wang, Shaoting Weng, Yajie Tang, Sen Lin, Xiayue Liu, Wenhui Zhang, Gang Liu, Boomi Pandi, Yinrong Wu, Lei Ma, Lin Wang
Cluster of differentiation 20 (CD20) is a nonglycosylated, multispanning transmembrane protein specifically integrated by B lymphocytes. Similar to CD20, another four-pass transmembrane protein, claudin 18.2, has attracted attention as an emerging therapeutic target for cancer. However, their poor solubility and toxic nature often hinder downstream applications, such as antibody drug development. Therefore
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An archaeal nitrile hydratase from the halophilic archaeon A07HB70 exhibits high tolerance to 3-cyanopyridine and nicotinamide Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-31 Junling Guo, Zhongyi Cheng, Zhemin Zhou
Nitrile hydratase (NHase, EC 4.2.1.84) is widely used in the industrial production of biosynthetic amide compounds. NHases obtained from prokaryotic and eukaryotic sources have been widely studied, while the NHases derived from archaeal sources have not been reported. Here, we focused on a distinctive NHase derived from a halophilic archaeon (archaeon A07HB70, A.r NHase) that thrives in high-salt environments
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Overexpression and truncation of a novel cold-adapted lipase with improved enzymatic characteristics Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-14 Yue Zhang, Yu Gao, Jiahui Chen, Fang Yu, Yongming Bao
The novel cold-adapted lipase (Lip ZC12) derived from Psychrobacter sp. ZY124 exhibited higher catalytic activity at 20–40 °C, the whole gene was then sequenced, analyzed, and overexpressed. However, its intrinsic structural characteristics lead to a decreased affinity toward the substrate, thus limiting the improvement of catalytic efficiency. Modeling the homologous structure and simulating the binding
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Recombinant expression and functional characterization of FadD2 protein in Mycobacterium tuberculosis Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-07 Dafeng Liu, Cai Yuan, Chenyun Guo, Mingdong Huang, Donghai Lin
Mycobacterium tuberculosis (Mtb) is a crucial and highly destructive intracellular pathogen responsible for causing tuberculosis (TB). The emergence and dissemination of multi-drug resistant Mtb has further aggravated the TB crisis, leading to high mortality. Mtb FadD2 is a fatty acyl-coenzyme A (CoA) synthetase that modifies the cell envelope and plays an important role in reducing Mtb susceptibility
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Production of enzymes for the removal of odorous substances in plant biomass Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-08 Patrick Opdensteinen, Matthias Knödler, Johannes F. Buyel
Residual plant biomass collected from agricultural, technical or biopharmaceutical processes contains odorous substances. The latter are often unacceptable for customers if the biomass is used in sustainable products such as building materials, paints, glues or flame-resistant foils. The objective of this study was to identify enzymes that can prevent the formation or facilitate the degradation of
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Uncovering the role of leucine 59 in Renilla luciferase stability and activity with error-prone PCR: Implications for protein engineering Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-08 Zahra Fanaei-kahrani, Rahman Emamzadeh, Mahboobeh Nazari
A new variant of Renilla luciferase, named Met C-SRLuc 8, was obtained from a random mutagenesis library and expressed in Escherichia coli BL21 (DE3) plys and purified. The results of the enzyme's binding affinity, kinetic stability, and bioinformatic studies demonstrated that leucine 59, located within the hot-spot foldon in the N-terminal domain of the protein, plays a significant role in the stability
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Antibody-mimetic drug conjugate with efficient internalization activity using anti-Her2 VHH and duocarmycin Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-04 Juri Sakata, Toshifumi Tatsumi, Akira Sugiyama, Akihiro Shimizu, Yuya Inagaki, Hiroto Katoh, Takefumi Yamashita, Kazuki Takahashi, Sho Aki, Yudai Kaneko, Takeshi Kawamura, Mai Miura, Masazumi Ishii, Tsuyoshi Osawa, Toshiya Tanaka, Shumpei Ishikawa, Masanobu Tsukagoshi, Michael Chansler, Tatsuhiko Kodamad, Motomu Kanai, Kenzo Yamatsugu
Antibody-mimetic drug conjugate (AMDC) is a cancer cell-targeted drug delivery system based on the non-covalent binding of mutated streptavidin and modified biotin, namely Cupid and Psyche. However, the development of AMDCs is hampered by difficulties in post-translational modification or poor internalization activity. Here, we report an expression, refolding, and purification method for AMDC using
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Efficient expression and purification of tag-free recombinant human procalcitonin (hPCT) with precise sequence in E. coli Protein Expres. Purif. (IF 1.6) Pub Date : 2023-10-02 Masataka Nakagawa, Yui Tomioka, Teruo Akuta
We present an efficient method for expression and purification of recombinant human procalcitonin (hPCT) in E. coli T7 express LysY/Iq cells, ensuring precise N- and C-terminal amino acid sequences. Our method involves fusing codon-optimized cDNA with two distinct tag sequences: eXact tag and chitin binding domain (CBD) tag. To purify the protein, we employ a two-step affinity chromatography process
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The construction of long-acting exendin-4 analog and its hypoglycemic effect in diabetic mice Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-18 Jingmin Cai, Linquan Zang, Xueman Wu, Zhiwen Liang, Ke Zheng, Lin Zhao, Huangjin Li
Exendin-4 is a glucagon-like polypeptide-1 (GLP-1) analog derived from lizard venom, but its short half-life affects drug administration compliance. An anti-HSA nanobody with a smaller size to guide the peptide coupling to Human Serum Albumin(HSA) in vivo may be a feasible strategy for constructing inexpensive, long-acting exendin-4 analogs. For this purpose, a fusion protein (exendin-4-(G4S)3-sdAbHSA)
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Insight into the thrombolytic ability of an extracellular fibrinolytic enzyme from Bacillus amyloliquefaciens GXU-1 isolated from Sipunculus nudus Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-12 Hongrui Jiang, Ruiyuan Shi, Xiaomei Li, Jiale Tang, Douyong Min
Bacterial fibrinolytic enzymes have an important role in thrombolytic therapy due to their ability to dissolve fibrin. Therefore, purification, characterization and activity determination are of prime importance for bacterial fibrinolytic enzymes. In the current study, marine Bacillus amyloliquefaciens was first screened from Sipunculus nudus living in the Beibu Gulf of China and denoted as Guangxi
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Generation and characterization of a monoclonal antibody against FGFR3 that protects mice from BoNT/A Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-13 Xianghua Xiong, Yujin Qiu, Jiahao Zheng, Ling Zhou, Qingyang Wang, Jinglun Pang, Weicai Zhang, Huipeng Chen, Gang Liu, Xiaodong Han
Botulinum neurotoxin serotype A (BoNT/A) can cause flaccid paralysis of muscles, an illness fatal to human, by entering neurons and blocking neurotransmitter release. The process was mediated by three receptors. A specific monoclonal antibody anti-D23, designated as ML419, targeting the ectodomain (D23) of fibroblast growth factor receptor 3 (FGFR3), one of the three receptors, was screened and capable
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Biochemical characterization of a novel bifunctional alginate lyase from Microbulbifer arenaceous Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-17 Jun Jiang, Zhengqiang Jiang, Qiaojuan Yan, Susu Han, Shaoqing Yang
Bio‒production of alginate oligosaccharides (AOSs), a type of functional food additive, is a promising way for green utilization of algae, in which alginate hydrolyzing enzymes play a key role. A novel alginate lyase gene (MiAly17A) from a marine bacterium Microbulbifer arenaceous was heterologously expressed in E. coli. The coding sequence of the gene shared the highest identity of 86% with a polysaccharide
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Optimized expression and purification of a human adenosine deaminase in E. coli and characterization of its Asp8Asn variant Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-07 Maria Rain Jennings, Soohyon Min, Grace S. Xu, Kassandra Homayuni, Bhavana Suresh, Yusef Amir Haikal, John Blazeck
Homo sapiens adenosine deaminase isoform 1 (HsADA1) hydrolyzes adenosine and 2-deoxyadenosine as a key step in the purine nucleoside salvage pathway. Some HsADA1 mutations have severe deleterious effects, as is the case in a severe combined immunodeficiency resulting from loss of enzyme activity (ADA-SCID). Other mutations that reduce enzyme activity, for instance the Asp8Asn (D8N) variant, do not
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Generation of a blockage monoclonal antibody of LILRB1 against HLA-G Protein Expres. Purif. (IF 1.6) Pub Date : 2023-09-06 Yunlong Shen, Ruirui Zhang, Xiaohua Jiang, Jinliang Yang
Leukocyte immunoglobulin like receptor B1 (LILRB1) is widely expressed in immune cells as an immunosuppressive receptor. Tumor cells highly express the ligand HLA-G, which inhibits the function of immune cells by binding to LILRB1, to achieve immune escape. LILRB1 is a potential immunotherapeutic target. This study developed a monoclonal antibody named B1M023 (B1M023 mAb) that could bind LILRB1 with
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Biochemical and biophysical characterization of the RAS family small GTPase protein DiRAS3 Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-29 Takashi Saitoh, Ha-Neul Kim, Riku Narita, Ibuki Ohtsuka, Weiyu Mo, Ki-Young Lee, Masahiro Enomoto, Geneviève M.C. Gasmi-Seabrook, Christopher B. Marshall, Mitsuhiko Ikura
DiRAS3, also called ARHI, is a RAS (sub)family small GTPase protein that shares 50–60% sequence identity with H-, K-, and N-RAS, with substitutions in key conserved G-box motifs and a unique 34 amino acid extension at its N-terminus. Unlike the RAS proto-oncogenes, DiRAS3 exhibits tumor suppressor properties. DiRAS3 function has been studied through genetics and cell biology, but there has been a lack
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Enhancement of recombinant human interleukin-22 production by fusing with human serum albumin and supplementing N-acetylcysteine in Pichia Pastoris Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-29 Yingqing Xu, Ziming Zhao, Zijian Geng, Hongwei Zhou, Chengxi Yang, Yixing Wang, Buayisham Kuerban, Yimeng Xiao, Gang Luo
Interleukin-22 (IL-22) plays an important role in the treatment of organ failure, which can induce anti-apoptotic and proliferative signaling pathways; Nevertheless, the practical utilization of IL-22 is hindered by the restricted efficacy of its production. Pichia pastoris presents a viable platform for both industrial and pharmaceutical applications. In this study, we successfully generated a fusion
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Purification and characterization of an asialofetuin specific lectin from the rhizome of Xanthosoma violaceum Schott Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-29 Oinam Sangita Devi, Senjam Sunil Singh, K. Rana, Sorokhaibam Jibankumar Singh, Wayenbam Sobhachandra Singh
Lectins are proteins or glycoproteins that bind specifically and reversibly to the carbohydrate or glycoconjugates. A new lectin is purified from the rhizome of Xanthosoma violaceum Schott. by successive steps of ammonium sulfate fractionation and affinity chromatography with asialofetuin as ligand. The purified lectin was found to be a homotetramer of approximately 49 kDa with a subunit molecular
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Production of recombinant human proliferating cellular nuclear antigen (PCNA) for structural and biophysical characterization Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-18 B. Vandborg, D.L. Holroyd, T. Pukala, J.B. Bruning
Human proliferating cell nuclear antigen (hPCNA) is a DNA replication processivity factor, which acts as a docking platform, allowing proteins to have access to the replication fork and increasing the affinity of DNA interacting proteins, making it critical for cell survival. The trimer forms a ring-shaped oligomer allowing DNA to pass through the middle and interacting proteins to dock on the outside
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Identification and characterization of a novel sativene synthase from Fischerella thermalis Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-23 Shu Chen, Jia Li, Ming Wang, Yuheng Liu, Tangli Li, Xiaopu Yin, Tian Xie, Rong Chen
Sesquiterpene synthases (TPS) determine the structural diversity of terpenoids, which are species specific. In this study, we report a TPS from Fischerella thermalis (named as FtTPS), recombinantly expressed as a soluble protein in Escherichia coli BL21(DE3) strain. The FtTPS protein could catalyze the conversion of farnesyl pyrophosphate (FPP) to sativene, a kind of tricyclic sesquiterpene. The optimal
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Strategies for rapid production of crystallization quality coatomer WD40 domains Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-23 Debajit Dey, S. Saif Hasan
The vesicular secretion of soluble cargo proteins from the endoplasmic reticulum (ER) is accompanied by the export of ER-resident membrane proteins that are co-packaged in secretory vesicles. The cytosolic coatomer protein complex I (COPI) utilizes the N-terminal WD40 domains of α-COPI and β′-COPI subunits to bind these membrane protein “clients” for ER retrieval. These “αWD40” and “β'WD40” domains
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The effect of the bgs13 mutation on the structure of the reporter protein beta-lactoglobulin: Influence on folding and aggregation in Pichia pastoris Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-19 Bushra Irshad, Cai Lu, Anneroos E. Nederstigt, Priscilla Hu, Liang Xue, Joan Lin-Cereghino, Andreas H. Franz, Joseph S. Harrison, Geoff P. Lin-Cereghino
Pichia pastoris, a methylotrophic yeast used for recombinant protein expression, has the capability of performing many eukaryotic post-translational modifications, growing to high cell densities, and producing proteins in a cost-effective manner. However, P. pastoris's secretion properties are not always efficient, and its secretory pathway mechanisms have not been thoroughly elucidated. A previously
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Highly efficient overexpression and purification of multisubunit tethering complexes in Saccharomyces cerevisiae Protein Expres. Purif. (IF 1.6) Pub Date : 2023-08-20 Qiushuang Ji, Ke Zhang, Na Cao, Xiaoyu You, Shuaihua Cao, Mengya Wang, Jiatian Guo, Hongwei Wang, Kunrong Mei
Vesicle trafficking is a fundamental cellular process that ensures proper material exchange between organelles in eukaryotic cells, and multisubunit tethering complexes (MTCs) are essential in this process. The heterohexameric homotypic fusion and protein sorting (HOPS) complex, which functions in the endolysosomal pathway, is a member of MTCs. Despite its critical role, the complex composition and